Our long-term goal is to develop highly effective natural killer (NK) cell-based immunotherapy for high-risk neuroblastoma, especially disease in primary and metastatic sites that may be multi-drug and/or radiation resistant. Our in vitro data strongly suggests that NK cells that have been activated by IL-2 and/or IL-15 (A-NK) can have a major anti-tumor effect. We hypothesize that the anti-tumor efficacy of NK cell-mediated immunotherapy can be improved 1) by increasing expression of NK chemokines in the tumor microenvironment to increase the quantity of infiltrating A-NK cells; and 2) by increasing tumor cell susceptibility to A-NK cell-mediated killing. If these hypotheses are correct, multiagent therapy that increases NK infiltration, survival, and function in the tumor microenvironment and that enhances sensitivity of tumor cells will maximize destruction of primary and metastatic disease in vivo. The following Specific Aims will test these hypotheses: 1) Increase NK infiltration and anti-tumor activity in microenvironments of adrenal and metastatic neuroblastoma. 2) Increase neuroblastoma susceptibility to direct A-NK cytotoxicily and to antibody dependent cellular (NK) cytotoxicity (ADCC) in vitro and in NOD/SCID mice. Research Design and Methods. The first aim will define chemokines for NK cells within the neuroblastoma microenvironment and determine if cytokines (IL-2, IL- 12, IL-18 alone and in combination) and an immunocytokine (anti-GD2 monoclonal antibody/IL-2 fusion protein) alone and in combination with IL-12 and IL18 enhance chemokine expression, NK infiltration, and tumor killing. NKT and NK cells, which secrete chemokines, will also be tested for their ability to recruit NK cells into the tumor microenvironment and increase killing. The second aim will define targets for NK cells expressed by neuroblastoma cells (NKG2D ligands that initiate perforin-dependent cytotoxicity and the death receptor TRAIL-R2) and will identify agents that increase cytotoxicity through these pathways. Agents to be tested include those that increase intracellular ceramide and reactive oxygen species to enhance perforin-dependent cytotoxicity and that inhibit hislone deacetylase and proteosomes to increase TRAIL-mediated killing. This research uses purified and well-characterized human NK and NKT cells and a unique panel of human neuroblastoma cell lines for in vitro and in vivo experiments. Neuroblastomas and bone marrow metastases from patients also are studied to guide and validate model experiments. Direct NK cytotoxicity and ADCC are measured in vitro with calcein-AM/DIMSCAN microcytotoxicity and flow cytometry assays. Therapy for NOD/SCID mice bearing adrenal and metastatic neuroblastomas uses adoptively transferred human NK cells together with agents that enhance their localization and function in the tumor microenvironment. Summary. The proposed research will provide a foundation for designing novel clinical trials for children with high-risk neuroblastoma. The orincioles that we establish should be aenerallv aoolicable to other childhood and adult malionancies.